This invention concerns functional K-252a derivatives that potentiate the activity of neurotrophins.
The etiology of neurodegenerative disorders is unknown. Such disorders may involve neurotrophins, which are low molecular weight polypeptides that play a role in the development, function, or survival of responsive cells.
Nerve growth factor (NGF) is the best characterized neurotrophin and is required for normal development and function of certain sensory, and cholinergic neurons (Levi-Montalcini, Annu. Rev. Neurosci. 5:341-362, 1982). Less is known about other neurotrophins, such as neurotrophin-3 (NT3), due in part to inadequate knowledge concerning the structure, function or binding properties of NT-3 responsive cell receptors. NT-3 plays a role in the survival and function of cholinergic neurons in the basal forebrain (Knusel et al., J. Neurochem. 59:715-722, 1992) and hippocampal neurons (Collazo et. al., Neuron 9:643-656, 1992), and, like NGF, may influence the survival or function of many cell types. NT-3, among other neurotrophins, exhibits characteristic patterns of activity in the nervous system (Maisonpierre et al., Science 247:1446-1451, 1991) which depends, in part, on productive cell receptor interactions.
The high affinity neurotrophic receptors, trks, comprise a family of proteins consisting of trk A, trk B. and trk C (see Knusel et al., supra). Members of this receptor family are membrane associated proteins that exhibit tyrosine kinase activity. Interaction of a neurotrophin ligand with trks induces the phosphorylation of specific tyrosine residues on the receptor. Phosphorylation of trks is an immediate response to neurotrophin binding. It is an absolute requirement for the activation of enzymatic pathways regulating functional responses to the neurotrophins by the cell (Klein et al., Cell 65:189-197, 991; Lamballe et al., Cell 66:967-979, 1991).
Ethical (implanting fetal cells producing endogenous and/or recombinant neurotrophin) and technical (ability to produce large quantities of pure neurotrophin) considerations limit the full therapeutic value of neurotrophins in the treatment of neurological disease. These considerations may prevent widespread use of NT-3 or other neurotrophins. Thus, new molecules that potentiate the neurotrophin activity are of special interest.
K-252a is an indolocarbazole alkaloid that was originally isolated from a Nocardiopsis sp. culture (Kase et al., J. Antibiotics 39:1059-1065, 1986). It is an inhibitor of several enzymes, including protein kinase C (Kase et al., Biochem. Biophys. Res. Comm. 142:436-440, 1987; Nakanishi et al., J. Biol. Chem. 263:6215-6219, 1988), and trk (Berg et al., J. Biol. Chem. 267:13-16, 1992). Consistent with this latter effect, K-252a blocks NGF mediated cell survival in some in vitro cell assays (Koizumi et al., J. Neurosci. 8: 715-721, 1988; Doherty et al., Neurosci. Lett. 96:1-6, 1989; Matsuda et al., Neurosci. Lett. 87:11-17, 1988), but not in other assay systems (Borasio, Neurosci. Lett. 108:207-212, 1990). K-252a will induce neurotrophin-like effects in certain neuronal cell types, but the chemically related K-252b will not (see Knusel et al. supra). These findings imply that multiple proteins interact with K-252a (Coussens et al., Science 233:859 -866, 1986). It is likely that such complex interactions are responsible for the conflicting data found in the prior art.
K-252a and K-252b influence the phosphorylation state of trks. For example, K-252a may inhibit neurotrophin receptor phosphorylation (Squinto et al. WO 93/00909). However, in some settings, K-252a and K-252b potentiate target cell actions and trk tyrosine phosphorylation mediated by NT-3 (Knusel et. al. supra). K-252a may be a non-competitive partial agonist of trks (Lewis et al., Society for Neuroscience, Abstract, 1:13, 1992).
The proposed uses of K-252a or related K-252a derivatives such as staurosporine include tumor inhibition (Murakata et al., U.S. 4,877,776; Nomoto et al. EPO 238,011; Caravatti et al., U.S. 5,093,330;), anti-insecticidal activity (McCoy et al., U.S. 4,735,939), and inhibition of inflammation (Bell et al., U.S. Pat. No. 4,816,450).